Fertilization involves a series of signal transduction events that serve to activate the block to polyspermy, induce resumption of the cell cycle, incorporate the paternal genome, and activate egg metabolism. Key steps in this process include the actin cytoskeleton modifications that physically incorporate the sperm nucleus into the egg and subsequently reorganize the egg cortex. Recent findings by this laboratory revealed that the cytoskeleton-associated PYK2 kinase is rapidly activated during fertilization and functional studies indicate a role for PYK2 in sperm incorporation and egg activation. The objective of the proposed study is to test the hypothesis that PYK2 kinase participates in the signaling events triggered by sperm-egg adhesion or fusion that are critical for sperm incorporation and modification of the egg cytoskeleton. The project will make use of the economical zebrafish fertilization system which provides unique advantages for identification of signaling events at the point of sperm-egg contact as well as for proteomic analysis of substrates of the PYK2 kinase. The first specific aim will identify the specific function(s) of PYK2 kinase in egg activation. The second aim will identify the signals which activate PYK2 at fertilization. The third aim will identify the protein targets phosphorylated by PYK2 in order to work out the downstream pathways that it controls. The results of this study will provide important details regarding control of the egg cytoskeleton at fertilization and possibly help resolve the question of signaling pathways activated by sperm-egg recognition and fusion. These findings will have significant application to the development of techniques for assisted reproductive technologies to enhance human fertility. Lay Summary: This study will work out the mechanism by which the sperm activates the egg during fertilization. The specific role of PYK2 kinase in bringing the sperm nucleus into the egg will be analyzed with the ultimate aim of enhancing the success of human in vitro fertilization.